Genomic Variability of Mycobacterium tuberculosis Strains of the Euro-American Lineage Based on Large Sequence Deletions and 15-Locus MIRU-VNTR Polymorphism

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RD^Rio deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the “Cameroon” family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification.     

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.     

Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains

Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

Mycobacterium tuberculosis lineage influences innate immune response and virulence and is associated with distinct cell envelope lipid profiles

The six major genetic lineages of Mycobacterium tuberculosis are strongly associated with specific geographical regions, but their relevance to bacterial virulence and the clinical consequences of infection are unclear. Previously, we found that in Vietnam, East Asian/Beijing and Indo-Oceanic strains were significantly more likely to cause disseminated tuberculosis with meningitis than those from the Euro-American lineage. To investigate this observation we characterised 7 East Asian/Beijing, 5 Indo-Oceanic and 6 Euro-American Vietnamese strains in bone-marrow-derived macrophages, dendritic cells and mice. East Asian/Beijing and Indo-Oceanic strains induced significantly more TNF-α and IL-1β from macrophages than the Euro-American strains, and East Asian/Beijing strains were detectable earlier in the blood of infected mice and grew faster in the lungs. We hypothesised that these differences were induced by lineage-specific variation in cell envelope lipids. Whole lipid extracts from East Asian/Beijing and Indo-Oceanic strains induced higher concentrations of TNF-α from macrophages than Euro-American lipids. The lipid extracts were fractionated and compared by thin layer chromatography to reveal a distinct pattern of lineage-associated profiles. A phthiotriol dimycocerosate was exclusively produced by East Asian/Beijing strains, but not the phenolic glycolipid previously associated with the hyper-virulent phenotype of some isolates of this lineage. All Indo-Oceanic strains produced a unique unidentified lipid, shown to be a phenolphthiocerol dimycocerosate dependent upon an intact pks15/1 for its production. This was described by Goren as the 'attenuation indictor lipid' more than 40 years ago, due to its association with less virulent strains from southern India. Mutation of pks15/1 in a representative Indo-Oceanic strain prevented phenolphthiocerol dimycocerosate synthesis, but did not alter macrophage cytokine induction. Our findings suggest that the early interactions between M. tuberculosis and host are determined by the lineage of the infecting strain; but we were unable to show these differences are driven by lineage-specific cell-surface expressed lipids.     

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.     

Genetic distinctions among clinical and environmental strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Presence of RD149 deletions in M. tuberculosis Central Asian Strain 1 isolates affect growth and TNFα induction in THP-1 monocytes

Central Asian Strain 1 (CAS1) is the prevalent Mycobacterium tuberculosis genogroup in South Asia. CAS1 strains carry deletions in RD149 and RD152 regions. Significance of these deletions is as yet unknown. We compared CAS1 strains with RD149 and concurrent RD149-RD152 deletions with CAS1 strains without deletions and with the laboratory reference strain, M. tuberculosis H37Rv for growth and for induction of TNFα, IL6, CCL2 and IL10 in THP-1 cells. Growth of CAS1 strains with deletions was slower in broth (RD149; p?=?0.024 and RD149-RD152; p?=?0.025) than that of strains without deletions. CAS1 strains with RD149 deletion strains further showed reduced intracellular growth (p?=?0.013) in THP-1 cells as compared with strains without deletions, and also as compared with H37Rv (p?=?0.007) and with CAS1 RD149-RD152 deletion strains (p?=?0.029). All CAS1 strains induced higher levels of TNFα and IL10 secretion in THP-1 cells than H37Rv. Additionally, CAS1 strains with RD149 deletions induced more TNFα secretion than those without deletions (p?=?0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid growth and induced lower levels of TNFα and IL6 secretion in THP-1 cells than isolates from pulmonary sources. This data suggests that presence of RD149 reduces growth and increases the induction of TNFα in host cells by CAS1 strains. Differences observed for extrapulmonary strains may indicate an adaptation which increases potential for dissemination and tropism outside the lung. Overall, we hypothesise that RD149 deletions generate genetic diversity within strains and impact interactions of CAS1 strains with host cells with important clinical consequences.     

Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10, and R11 potato lines.

In Phytophthora infestans, a cluster of three dominant avirulence genes is located on the distal part of linkage group VIII. In a mapping population from a cross between two Dutch field isolates, probe M5.1, derived from an amplified fragment length polymorphism (AFLP) marker linked to the Avr3-Avr10-Avr11 cluster, hybridized only to DNA from the parent and F1 progeny that is avirulent on potato lines carrying the R3, R10, and R11 resistance gene. In the virulent parent and the virulent progeny, no M5.1 homologue was detected, demonstrating a deletion on that part of linkage group VIII. P. infestans is diploid, so the avirulent strains must be hemizygous for the region concerned. A similar situation was found in another mapping population from two Mexican strains. The deletion was also found to occur in many field isolates. In a large set of unique isolates collected in The Netherlands from 1980 to 1991, 37% had no M5.1 homologue and the deletion correlated strongly with gain of virulence on potato lines carrying R3, R10, and R11. Also, in some old isolates that belong to a single clonal lineage (US-1) and are thus highly homogenous, deletions at the M5.1 locus were detected, indicating that this region is unstable.     

Host- and tissue-specific pathogenic traits of Staphylococcus aureus

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.     

Mycobacterium tuberculosis Strains Potentially Involved in the TB Epidemic in Sweden a Century Ago

A hundred years ago the prevalence of tuberculosis (TB) in Sweden was one of the highest in the world. In this study we conducted a population-based search for distinct strains of Mycobacterium tuberculosis complex isolated from patients born in Sweden before 1945. Many of these isolates represent the M. tuberculosis complex population that fueled the TB epidemic in Sweden during the first half of the 20^th century.

Methods

Genetic relationships between strains that caused the epidemic and present day strains were studied by spoligotyping and restriction fragment length polymorphism.

Results

The majority of the isolates from the elderly population were evolutionary recent Principal Genetic Group (PGG)2/3 strains (363/409 or 88.8%), and only a low proportion were ancient PGG1 strains (24/409 or 5.9%). Twenty-two were undefined. The isolates demonstrated a population where the Euro-American superlineage dominated; in particular with Haarlem (41.1%) and T (37.7%) spoligotypes and only 21.2% belonged to other spoligotype families. Isolates from the elderly population clustered much less frequently than did isolates from a young control group population.

Conclusions

A closely knit pool of PGG2/3 strains restricted to Sweden and its immediate neighbours appears to have played a role in the epidemic, while PGG1 strains are usually linked to migrants in todaýs Sweden. Further studies of these outbreak strains may give indications of why the epidemic waned.     

The population structure of Mycobacterium tuberculosis in Mongolia as assessed using large sequence polymorphism analysis

112 strains of M. tuberculosis isolated from lung tuberculosis patients in Mongolia were genotyped using RD9, RD7, TbD1, RD105, and RD750 loci. The genotypes of all the strains studied were characterized using the conservation of RD7, RD9, and RD750 loci and the presence of the deletion in the locus TbD1. RD105 was detected in 65 isolates (58%). The isolate was classified into two groups--East-Asian and Euro-American.     

VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.     

Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method

Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR-PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time.     

Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.     

Genetic Distinctions among Clinical and Environmental Strains of Vibrio vulnificus

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n = 35) or clinical (n = 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical “clinical” or “environmental” polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the “environmental” profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical “clinical” profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the “clinical” polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.